Semaphorins were discovered in the early 1990's as repulsive molecules for neuronal growth cones (Non Patent Literatures 1 and 2). More than 20 members of semaphorins have been identified to date, and the semaphorin family is characterized by sharing a conserved region consisting of about 500 amino acids, referred to as the Sema domain. These semaphorins are classified into eight sub-classes (Sema 1-7, V) on the basis of structural difference at the C-terminus following the Sema domain.
Plexins (Plexin-A1/A2/A3/A4, Plexin-B1/B2/B3, Plexin-C1, and Plexin-D1) and neuropilins (Nrp-1 and Nrp-2) are known as major receptors that contribute to the semaphorin activity. Semaphorins are known to also bind to integrins, CD72, and Tim-2 (Non Patent Literature 3). Furthermore, the semaphorin receptors, plexins, are known to associate with various co-receptors such as VEGFR-2, c-Met, and Trem2/DAP12, and construct complex ligand-receptor relationships, which possibly reflect diverse functions of the semaphorins. Indeed, the semaphorins have diverse biological activities such as angiogenesis/vasculogenesis, cancer metastasis/invasion, bone metabolism regulation, retinal homeostasis, and immune regulation, and the involvement of the semaphorins in various diseases such as allergic diseases or autoimmune diseases, metabolic bone diseases, neurodegenerative diseases, retinitis pigmentosa, sudden death from cardiac causes, and cancer metastasis/invasion has been successively reported in the past several years (Non Patent Literature 3). In connection with biological activities of the semaphorins, active research is currently ongoing with a view towards developing diagnostic/therapeutic methods for human diseases.
Plexin-A1 is a receptor for class III and class VI semaphorins. Plexin-A1 has been reported to form receptors together with VEGF receptor and Off-Track during chick heart morphogenesis, and also act as a receptor for neuronal repulsive factors by forming a receptor for class III semaphorins together with Nrp-1. Furthermore, Plexin-A1 has been reported to also act as a receptor for class VI semaphorins, Sema6C and Sema6D, and be involved in axon guidance or cardiac organogenesis.
It has been reported that suppression of Plexin-A1 expression by shRNA in mouse dendritic cells, for example, leads to attenuated non-autoimmune T-cell immunity in vivo or in vitro (Non Patent Literature 4). Plexin-A1 signal analysis in dendritic cells and osteoclasts has also confirmed that Plexin-A1 forms heteroreceptors with Trem-2 and DAP-12 in these cells. It has also been shown that recombinant soluble Sema6D protein stimulation promotes the expression of inflammatory cytokines such as IL-12 from dendritic cells and osteoclast differentiation from precursor cells, and that while Sema6D binds to wild-type dendritic cells, it hardly binds to dendritic cells from Plexin-A1-deficient mice. It has been reported that T-cell immune responses are significantly weaker in Plexin-A1-deficient mice, which spontaneously develops osteopetrosis-like symptoms caused by abnormal osteoclast differentiation (Non Patent Literature 5). Plexin-A1 inhibition by shRNA in mouse dendritic cells has shown that Plexin-A1 controls actin cytoskeleton localization in the immune synapses of dendritic cells and T cells via activation of signal transduction factor Rho (Non Patent Literature 6).
Furthermore, it has been reported that Plexin-A1 is involved in the migration of dendritic cells to the lymph nodes and in antigen-specific T-cell responses. It has also been reported that the expression of Sema3A, rather than Sema6C or Sema6D, is required for migration of dendritic cells as they pass through the endothelial cells of the lymphatic vessels, and Sema3A stimulates myosin-II activity and induce actomyosin contraction (Patent Literature 1).
Furthermore, in connection with Plexin-A2, which is another molecule belonging to the Plexin-A family, a low-resolution (7.0 A) structure of a triple complex, “Sema3A-Plexin-A2-Nrp-1”, has been disclosed. Although the binding between Sema3A and Plexin-A2 is too weak to be detected, it has been reported that an interaction between Sema3A and Plexin-A2 was detected in the presence of Nrp-1, even though it was far too weak for the expression of biological activity of Sema3A (Non Patent Document 7). The elucidation of this complex, however, requires further detailed study, because the resolution of this structure is extremely low, and a partial-length protein rather than a full-length protein was used. Non Patent Literature 8, on the other hand, introduces the structure of a triplet complex, “Sema3A-Plexin-A1-Nrp-1”, citing Non Patent Literature 7 described above. Non Patent Literature 7, however, fails to disclose the structure of the triplet complex “Sema3A-Plexin-A1-Nrp-1”.
Sema3A has been suggested to exhibit therapeutic effects against various diseases such as autoimmune diseases including pruritus due to psoriasis and atopic dermatitis, allergic rhinitis, osteoporosis, rheumatoid arthritis, and systemic lupus erythematosus, inflammatory diseases, and tumors, mainly through expression analysis in patients and experiments with animal models.
For example, decreased expression of Sema3A has been reported in the skin of patients with psoriasis or atopic dermatitis (Non Patent Literatures 9 and 10). Sema3A has inhibitory activity on C-fiber neurite outgrowth. In the skin of patients with psoriasis or atopic dermatitis, the decreased expression of Sema3A induces the outgrowth of C-fiber neurites, which is believed to result in susceptibility to itchiness. Indeed, the application of Sema3A to the skin of atopic dermatitis mouse models through intradermal administration or as an ointment has been reported to improve pruritic behavior caused by atopic dermatitis (Non Patent Literatures 11 and 12).
Sema3A has also been reported to be involved in airway hyperreactivity in allergic rhinitis, for example (Non Patent Literature 13). The expression of Sema3A decreases in epithelial cells in the nasal cavity of allergic rhinitis mouse models, which leads to an increased innervation density in the nasal turbinate lamina propria. This is believed to be one cause of the exacerbation of hypersensitivity such as sneezing and itching. Intranasal administration of Sema3A to allergic rhinitis mouse models has been reported to reduce the innervation density in the nasal turbinate lamina propria, and improve sneezing or pruritic behavior.
Furthermore, Sema3A is known to be also involved in the control of bone density. Sema3A has an activity to both activate osteoblasts and suppress osteoclast differentiation in vitro, and mice with systemic Sema3A deficiency develop osteoporosis-like symptoms. Furthermore, the administration of Sema3A to mouse models in which osteoporosis-like symptoms were induced by oophorectomy has been reported to improve the bone density (Non Patent Literature 14). In addition to the direct action of Sema3A upon osteoblasts and osteoclasts, a Sema3A-mediated bone density control mechanism through sensory innervation to bone has also been reported. In neuron-specific Sema3A-deficient mice, an osteoporosis-like decrease in bone density comparable to that in mice with systemic Sema3A deficiency has been reported (Non Patent Literature 5).
Furthermore, Sema3A has been reported to be involved in autoimmune diseases and inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus. For example, there is a report that, compared to normal human peripheral blood, the peripheral blood of rheumatoid arthritis patients shows a decrease in the level of Sema3A mRNA or protein produced upon in vitro activation of peripheral mononuclear blood cells (PBMCs), CD4-positive T cells, and CD8-positive T cells with anti-CD3 antibody and anti-CD28 antibody. Likewise, a decrease in the mRNA expression of Sema3A has been reported in rheumatoid arthritis patient-derived synovial tissue, compared to healthy human-derived synovial tissue. Furthermore, intraperitoneal administration of plasmids encoding Sema3A protein to collagen-induced arthritis mouse models has been reported to show an improvement in arthritis score or an improvement in hindlimb swelling (Non Patent Literature 16).
It has also been reported that the Sema3A concentration in the peripheral blood of systemic lupus erythematosus patients is significantly lower than that in the peripheral blood of healthy humans, and that in CD19+CD25high B cells collected from the peripheral blood of systemic lupus erythematosus patients, the expression of Sema3A is lower than that in the same cells collected from healthy individuals, which suggests the possibility that this change in the expression level of Sema3A in systemic lupus erythematosus patients may affect the activation of B cells (Non Patent Literature 17).
Furthermore, the expression of Sema3A has been reported to be lower in malignant melanoma skin tissues from melanoma patients than that in normal skin tissues. It has also been reported that murine malignant melanoma cell lines in which the Sema3A gene was transfected and stably expressed exhibit reduced migration and invasiveness of tumor cells in vitro and increased sensitivity to anti-cancer agents, compared to parental cell lines not transfected with the Sema3A gene. Likewise, in mouse models injected subcutaneously with tumor cells, murine malignant melanoma cell lines in which the Sema3A gene was transfected and stably expressed have been reported to exhibit suppressed tumor metastasis and retarded tumor growth, compared to parental cell lines not transfected with the Sema3A gene (Non Patent Literature 18).
These reports have suggested the utility of Sema3A as a therapeutic drug for various diseases such as autoimmune diseases including pruritus due to psoriasis and atopic dermatitis, allergic rhinitis, osteoporosis, rheumatoid arthritis, and systemic lupus erythematosus, inflammatory diseases, and tumors.
However, no report has heretofore been made on an antibody that binds to one of the receptors, Plexin-A1, and has an activity similar to that of Sema3A (agonist antibody). Furthermore, it has been unclear as to which region of the amino acid sequence of Plexin-A1 is bound by the antibody that binds to Plexin-A1 and has agonistic activity.